This project focuses on glycoconjugates of Schwann cells and oligodendrocytes during myelination and demyelination. A major aspect of the research concerns the myelin-associated glycoprotein (MAG), which is localized in periaxonal glial membranes of myelinated fibers and functions in transmitting signals between axons and myelin-forming cells. MAG is in the siglec subgroup of the immunoglobulin superfamily and binds to glycoconjugates containing terminal alpha2-3-linked sialic acid, suggesting that its axonal receptor or ligand could be a glycoprotein or ganglioside. In the past year, there have four reports of potential physiological binding partners for MAG, which include our own finding of a novel glycosylated isoform of microtubule associated protein 1B on the neuronal surface, GD1a and GT1b gangliosides, a p75 neurotrophin receptor/GT1b ganglioside complex and the Nogo receptor. Previous studies from our laboratory and others on MAG-null mice indicate that the most important functions of MAG are different in the CNS and PNS. In the CNS, its primary role appears to be in signaling from axons to oligodendrocytes to promote myelin formation and oligodendroglial health. However, in the PNS, it is essential for signaling in the opposite direction from Schwann cells to axons that is needed for the normal maintenance of myelinated axons. In the absence of MAG, the pathology in the PNS is characterized by axonal degeneration in association with a reduction of axonal caliber caused by cytoskeletal abnormalities including decreased expression and phosphorylation of neurofilaments. We have shown that this decreased phosphorylation of neurofilaments in MAG-null mice is due in part to decreased activities of extracellular signal regulated kinases (ERKs) and cyclin dependent kinase 5 (cdk5). The explanation for this PNS pathology in vivo could be either that MAG itself is part of a signal transduction pathway that is necessary for axonal maintenance or that there is a general breakdown of the Schwann cell-axon junction in the absence of MAG that disrupts signaling to the axon by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. In these experiments, neurons were co-cultured with COS-7 cells stably transfected with MAG or treated with a soluble MAG Fc-chimera. The presence of MAG caused elevated ERK 1/2 and cdk5 activities and increased expression and phosphorylation of neurofilament subunits and other cytoskeletal proteins, thereby supporting the direct involvement of MAG in the signaling pathway. It is hypothesized that the well-known role of MAG as a prominent white matter component that inhibits axonal growth is due to the physiologically important MAG-mediated signaling, which promotes the stability of mature myelinated axons, being received inappropriately by plastic regenerating axons in vivo or developing neurites in vitro. In addition, we have continued to use these in vitro models for pharmacological investigation of molecular mechanisms of MAG-mediated signal transduction in neurons. These studies demonstrate that inhibitors of adenylyl cyclase enhance the increased phosphorylation of cytoskeletal elements caused by MAG, suggesting that c-AMP down regulates the effect of MAG. Also, the effects of MAG were abolished by inhibition of phosphatidylinositol-3-kinase (PI3K), indicating that the signaling pathway involves or is modulated by PI3K. We are also using these in vitro models to determine if any of the potential MAG binding partners described above serves as the MAG receptor for the signaling that affects the axonal cytoskeleton. Previous morphological studies on the CNS of MAG-null mice revealed aberrant or redundant myelin loops, supernumerary myelin sheaths, abnormal paranodal structures and a significant delay of myelination. Thus in the absence of MAG, some oligodendrocytes do not seem to be efficient at determining when, where and how much myelin to form. Furthermore, there are a loss of oligodendroglial proteins and degeneration of periaxonal oligodendroglial processes in aging MAG-null mice, consistent with a dying back oligodendrogliopathy similar to that occurring in some demyelinating lesions of multiple sclerosis. These findings suggest that MAG functions as the receptor for an axonal signal that enhances the process of myelination and the vitality of oligodendrocytes. Experiments are in progress with primary oligodendrocyte cultures and oligodendroglial cell lines to test for and characterize the putative MAG-mediated signaling pathway that promotes the differentiation of oligodendrocytes.